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Studijn í obor Bioinformatika. last lecture summary. Meiosis. studying genomes. Studying DNA. Enzymes for DNA manipulation. Before 1970s, the only way in which individual genes could be studied was by classical genetics. - PowerPoint PPT Presentation
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Studijní obor Bioinformatika
LAST LECTURE SUMMARY
Meiosis
STUDYING GENOMES
Studying DNA
Enzymes for DNA manipulation• Before 1970s, the only way in which individual genes
could be studied was by classical genetics.• Biochemical research provided (in the early 70s)
molecular biologists with enzymes that could be used to manipulate DNA molecules in the test tube.
• Molecular biologists adopted these enzymes as tools for manipulating DNA molecules in pre-determined ways, using them to make copies of DNA molecules, to cut DNA molecules into shorter fragments, and to join them together again in combinations that do not exist in nature.
• These manipulations form the basis of recombinant DNA technology.
Recombinant DNA technology• The enzymes available to the molecular biologist fall into
four broad categories:1. DNA polymerase – synthesis of new polynucleotides
complementary to an existing DNA or RNA template2. Nucleases – degrade DNA molecules by breaking the
phosphodiester bonds• restriction endonucleases (restriction enzyme) – cleave DNA
molecules only when specific DNA sequences is encountered3. Ligases – join DNA molecules together4. End modification enzymes – make changes to the ends of
DNA molecules
source: Brown T. A. , Genomes. 2nd ed. http://www.ncbi.nlm.nih.gov/books/NBK21129/
DNA cloning• DNA cloning (i.e. copying) – logical extension of the ability
to manipulate DNA molecules with restriction endonucleases and ligases
• vector• DNA sequence that naturally replicates inside bacteria.• It consists of an insert (transgene) and larger sequence serving
as the backbone of the vector.• Used to introduce a specific gene into a target cell. Once the
expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation machinery ribosomal complexes.
Vectors• plasmid
• DNA molecule that is separated from, and can replicate independently of, the chromosomal DNA.
• Double stranded, usually circular, occurs naturally in bacteria.• Serves as an important tool in genetics and biotechnology labs,
where it is commonly used to multiply (clone) or express particular genes.
• length of insert: 1-10 kbp
source: wikipedia
Vectors• BAC (bacterial artificial chromosome)
• It is a particular plasmid found in E. coli. A typical BAC can carry about 250 kbp (100-350 kbp).
• cosmid • 40-45 kbp
• YAC (yeast artificial chromosome)• 1.5-3.0 Mbp
source: Brown T. A. , Genomes. 2nd ed. http://www.ncbi.nlm.nih.gov/books/NBK21129/
restriction endonuclease
ligase
DNA cloning
PCR – Polymerase chain reaction• DNA cloning results in the purification of a single fragment
of DNA from a complex mixture of DNA molecules.• Major disadvantage: it is time-consuming (several days to
produce recombinants) and, in parts, difficult procedure.• The next major technical breakthrough (1983) after gene
cloning was PCR.• It achieves the amplifying of a short fragment of a DNA
molecule in a much shorter time, just a few hours.• PCR is complementary to, not a replacement for, cloning
because it has its own limitations: we need to know the sequence of at least part of the fragment.
Mapping genomes
What is it about?• Assigning/locating the specific gene to the particular
region at the chromosome and determining the location and relative distances between genes at the chromosome.
• There are two types of maps: • genetic linkage map – shows the arrangement of genes (or other
markers) along the chromosomes as calculated by the frequency with which they are inherited together
• physical map – representation of the chromosomes, providing the physical distance between landmarks on the chromosome, ideally measured in nucleotide bases• The ultimate physical map is the complete sequence itself.
Genetic linkage map• Constructed by observing how frequently two markers
(e.g. genes, but wait till next slides) are inherited together.• Two markers located on the same chromosome can be
separated only through the process of recombination.• If they are separated, childs will have just one marker
from the pair.• However, the closer the markers are each to other, the
more tightly linked they are, and the less likely recombination will separate them. They will tend to be passed together from parent to child.
• Recombination frequency provides an estimate of the distance between two markers.
Genetic linkage map• On the genetic maps distances between markers are measured
in terms of centimorgans (cM).• 1cM apart – they are separated by recombination 1% of the time
• 1 cM is ROUGHLY equal to physical distance of 1 Mbp in human
Value of genetic map – marker analysis
• Inherited disease can be located on the map by following the inheritance of a DNA marker present in affected individuals (but absent in unaffected individuals), even though the molecular basis of the disease may not yet be understood nor the responsible gene identified.
• This represent a cornerstone of testing for genetic diseases.
Genetic markers• A genetic map must show the positions of distinctive
features – markers.• Any inherited physical or molecular characteristic that
differs among individuals and is easily detectable in the laboratory is a potential genetic marker.
• Markers can be • expressed DNA regions (genes) or • DNA segments that have no known coding function but which
inheritance pattern can be followed. • genes – not ideal, larger genomes (e.g. vertebrates) →
gene maps are not very detailed (low gene density)
Genetic markers• Must be polymorphic, i.e. alternative forms (alleles) must
exist among individuals so that they are detectable among different members in family studies.
• Variations within exons (genes) – lead to observable changes (e.g. eye color)
• Most variations occur within introns, have little or no effect on an organism, yet they are detectable at the DNA level and can be used as markers.1. restriction fragment length polymorphisms (RFLPs)2. simple sequence length polymorphisms (SSLPs)3. single nucleotide polymorphisms (SNPs, pronounce “snips”)
RFLPs• Recall that restriction enzymes cut DNA molecules at specific
recognition sequences.• This sequence specificity means that treatment of a DNA
molecule with a restriction enzyme should always produce the same set of fragments.
• This is not always the case with genomic DNA molecules because some restriction sites exist as two alleles, one allele displaying the correct sequence for the restriction site and therefore being cut, and the second allele having a sequence alteration so the restriction site is no longer recognized.
source: Brown T. A. , Genomes. 2nd ed. http://www.ncbi.nlm.nih.gov/books/NBK21129/
SSLPs• Repeat sequences that display length variations, different alleles
contain different numbers of repeat units (i.e. SSLPSs are multi-allelic).
• variable number of tandem repeat sequences (VNTRs, minisatellites)• repeat unit up to 25 bp in length
• simple tandem repeats (STRs, microsatellites)• repeats are shorter, usually di- or tetranucleotide
source: Brown T. A. , Genomes. 2nd ed. http://www.ncbi.nlm.nih.gov/books/NBK21129/
SNPs• Positions in a genome where some individuals have one
nucleotide and others have a different nucleotide.• Vast number of SNPs in every genome.• Each SNP could have potentially four alleles, most exist in
just two forms.• The value of two-allelic marker (SNP, RFLP) is limited by
the high possibility that the marker shows no variability among the members of an interesting family.
• The advantages of SNP over RFLP:• they are abundant (human genome: 1.5 millions of SNPs, 100 000
RFLPs)• easire to type (i.e. easier to detect)
Genome maps
source: Talking glossary of genetic terms, http://www.genome.gov/glossary/
relative locations of genes are established by following inheritance
patterns
visual appearance of a chromosome when stained and examined under a
microscope
the order and spacing of the genes, measured in base pairs
more at http://www.informatics.jax.org/silver/chapters/7-1.shtml
sequence map